The thioredoxin TRX-1 modulates the function of the insulin-like neuropeptide DAF-28 during dauer formation in Caenorhabditis elegans

Thioredoxins comprise a conserved family of redox regulators involved in many biological processes, including stress resistance and aging. We report that the C. elegans thioredoxin TRX-1 acts in ASJ head sensory neurons as a novel modulator of the insulin-like neuropeptide DAF-28 during dauer formation. We show that increased formation of stress-resistant, long-lived dauer larvae in mutants for the gene encoding the insulin-like neuropeptide DAF-28 requires TRX-1 acting in ASJ neurons, upstream of the insulin-like receptor DAF-2. Genetic rescue experiments demonstrate that redox-independent functions of TRX-1 specifically in ASJ neurons are needed for the dauer formation constitutive (Daf-c) phenotype of daf-28 mutants. GFP reporters of trx-1 and daf-28 show opposing expression patterns in dauers (i.e. trx-1 is up-regulated and daf-28 is down-regulated), an effect that is not observed in growing L2/L3 larvae. In addition, functional TRX-1 is required for the down-regulation of a GFP reporter of daf-28 during dauer formation, a process that is likely subject to DAF-28-mediated feedback regulation. Our findings demonstrate that TRX-1 modulates DAF-28 signaling by contributing to the down-regulation of daf-28 expression during dauer formation. We propose that TRX-1 acts as a fluctuating neuronal signaling modulator within ASJ neurons to monitor the adjustment of neuropeptide expression, including insulin-like proteins, during dauer formation in response to adverse environmental conditions.     

The thioredoxin TRX-1 regulates adult lifespan extension induced by dietary restriction in Caenorhabditis elegans

Dietary restriction (DR) is the only environmental intervention known to extend adult lifespan in a wide variety of animal models. However, the genetic and cellular events that mediate the anti-aging programs induced by DR remain elusive. Here, we used the nematode Caenorhabditis elegans to provide the first in vivo evidence that a thioredoxin (TRX-1) regulates adult lifespan extension induced by DR. We found that deletion of the gene trx-1 completely suppressed the lifespan extension caused by mutation of eat-2, a genetic surrogate of DR in the worm. However, trx-1 deletion only partially suppressed the long lifespan caused by mutation of the insulin-like receptor gene daf-2 or by mutation of the sensory cilia gene osm-5. A trx-1::GFP translational fusion expressed from its own promoter in ASJ neurons (Ptrx-1::trx-1::GFP) rescued the trx-1 deletion-mediated suppression of the lifespan extension caused by mutation of eat-2. This rescue was not observed when trx-1::GFP was expressed from the ges-1 promoter in the intestine. In addition, overexpression of Ptrx-1::trx-1::GFP extended lifespan in wild type, but not in eat-2 mutants. trx-1 deletion almost completely suppressed the lifespan extension induced by dietary deprivation (DD), a non-genetic, nutrient-based model of DR in the worm. Moreover, DD upregulated the expression of a trx-1 promoter-driven GFP reporter gene (Ptrx-1::GFP) in ASJ neurons of aging adults, but not that of control Pgpa-9::GFP (which is also expressed in ASJ neurons). We propose that DR activates TRX-1 in ASJ neurons during aging, which in turn triggers TRX-1-dependent mechanisms to extend adult lifespan in the worm.     

Spermatocyte/spermatid-specific thioredoxin-3, a novel Golgi apparatus-associated thioredoxin, is a specific marker of aberrant spermatogenesis

Mammalian germ cells are endowed with a complete set of thioredoxins (Trx), a class of redox proteins located in specific structures of the spermatid and sperm tail. We report here the characterization, under normal and pathological conditions, of a novel thioredoxin with a germ line-restricted expression pattern, named spermatocyte/spermatid-specific thioredoxin-3 (SPTRX-3). The human SPTRX-3 gene maps at 9q32, only 50 kb downstream from the TRX-1 gene from which it probably originated as genomic duplication. Therefore, human SPTRX-3 protein comprises a unique thioredoxin domain displaying high homology with the ubiquitously expressed TRX-1. Among the tissues investigated, Sptrx-3 mRNA is found exclusively in the male germ cells at pachytene spermatocyte and round spermatid stages. Light and electron microscopy show SPTRX-3 protein to be predominately located in the Golgi apparatus of pachytene spermatocytes and round and elongated spermatids, with a transient localization in the developing acrosome of round spermatids. In addition, increased levels of SPTRX-3, possibly caused by overexpression, are observed in morphologically abnormal human spermatozoa from infertile men. In addition, SPTRX-3 is identified as a novel postobstruction autoantigen. In this report, we propose that SPTRX-3 can be used as a specific marker for diverse sperm and testis pathologies. SPTRX-3 is the first thioredoxin specific to the Golgi apparatus, and its function within this organelle might be related to the post-translational modification of proteins required for germ cell-specific functions, such as acrosomal biogenesis.     

Depletion of cytosolic or mitochondrial thioredoxin increases CYP2E1-induced oxidative stress via an ASK-1-JNK1 pathway in HepG2 cells

Thioredoxin is an important reducing molecule in biological systems. Increasing CYP2E1 activity induces oxidative stress and cell toxicity. However, whether thioredoxin protects cells against CYP2E1-induced oxidative stress and toxicity is unknown. SiRNA were used to knockdown either cytosolic (TRX-1) or mitochondrial thioredoxin (TRX-2) in HepG2 cells expressing CYP2E1 (E47 cells) or without expressing CYP2E1 (C34 cells). Cell viability decreased 40-60% in E47 but not C34 cells with 80-90% knockdown of either TRX-1 or TRX-2. Depletion of either thioredoxin also potentiated the toxicity produced either by a glutathione synthesis inhibitor or by TNFα in E47 cells. Generation of reactive oxygen species and 4-HNE protein adducts increased in E47 but not C34 cells with either thioredoxin knockdown. GSH was decreased and adding GSH completely blocked E47 cell death induced by either thioredoxin knockdown. Lowering TRX-1 or TRX-2 in E47 cells caused an early activation of ASK-1, followed by phosphorylation of JNK1 after 48 h of siRNA treatment. A JNK inhibitor caused a partial recovery of E47 cell viability after thioredoxin knockdown. In conclusion, knockdown of TRX-1 or TRX-2 sensitizes cells to CYP2E1-induced oxidant stress partially via ASK-1 and JNK1 signaling pathways. Both TRX-1 and TRX-2 are important for defense against CYP2E1-induced oxidative stress.     

Thioredoxin-2 (TRX-2) is an essential gene regulating mitochondria-dependent apoptosis

Thioredoxin-2 (Trx-2) is a mitochondria-specific member of the thioredoxin superfamily. Mitochondria have a crucial role in the signal transduction for apoptosis. To investigate the biological significance of Trx-2, we cloned chicken TRX-2 cDNA and generated clones of the conditional Trx-2-deficient cells using chicken B-cell line, DT40. Here we show that TRX-2 is an essential gene and that Trx-2-deficient cells undergo apoptosis upon repression of the TRX-2 transgene, showing an accumulation of intracellular reactive oxygen species (ROS). Cytochrome c is released from mitochondria, while caspase-9 and caspase-3, but not caspase-8, are activated upon inhibition of the TRX-2 transgene. In addition, Trx-2 and cytochrome c are co-immunoprecipitated in an in vitro assay. These results suggest that mitochondrial Trx-2 is essential for cell viability, playing a crucial role in the scavenging ROS in mitochondria and regulating the mitochondrial apoptosis signaling pathway.     

Selenocysteine-containing thioredoxin reductase in C. elegans.

Mammalian thioredoxin reductases contain a TGA-encoded C-terminal penultimate selenocysteine (Sec) residue, and show little homology to bacterial, yeast, and plant thioredoxin reductases. Here we show that the nematode, Caenorhabditis elegans, contains two homologs related to the mammalian thioredoxin reductase family. The gene for one of these homologs contains a cysteine codon in place of TGA, and its product, designated TR-S, was previously suggested to function as thioredoxin reductase. The other gene contains TGA and its product is designated TR-Se. This Sec-containing thioredoxin reductase lacks a canonical Sec insertion sequence element in the 3'-untranslated area of the gene. TR-Se shows greater sequence similarity to mammalian thioredoxin reductase isozymes TR1 and TR2, whereas TR-S is more similar to TR3. TR-Se was identified as a thioredoxin reductase selenoprotein by labeling C. elegans with 75Se and characterizing the resulting 75Se-labeled protein by affinity and other column chromatography and gel-electrophoresis. TR-Se was expressed in Escherichia coli as a selenoprotein when a bacterial SECIS element was introduced downstream of the Sec TGA codon. The data show that TR-Se is the major naturally occurring selenoprotein in C. elegans, and suggest an important role for selenium and the thioredoxin system in this organism.     

Functional characterization of thioredoxin 3 (TRX-3), a Caenorhabditis elegans intestine-specific thioredoxin

Thioredoxins are a class of evolutionarily conserved proteins that have been demonstrated to play a key role in many cellular processes involving redox reactions. We report here the genetic and biochemical characterization of Caenorhabditis elegans TRX-3, the first metazoan thioredoxin with an intestine-specific expression pattern. By using green fluorescent protein reporters we have found that TRX-3 is expressed in both the cytoplasm and the nucleus of intestinal cells, with a prominent localization at the apical membrane. Although intestinal function, reproductive capacity, longevity, and resistance of trx-3 loss-of-function mutants to many stresses are indistinguishable from those of wild-type animals, we have observed a slight reduction in size and a minor reduction in the defecation cycle timing of trx-3 mutants. Interestingly, trx-3 is induced upon infection by Photorhabdus luminescens and Candida albicans, and TRX-3 overexpression provides a modest protection against these pathogens. Together, our data indicate that TRX-3 function in the intestine is dispensable for C. elegans development but may be important to fight specific bacterial and fungal infections.     

Some highlights of research on aging with invertebrates, 2008

This annual review focuses on invertebrate model organisms, which shed light on new mechanisms in aging and provide excellent systems for in-depth analysis. This year, the first quantitative estimate of evolutionary conservation of genetic effects on lifespan has pointed to the key importance of genes involved in protein synthesis, a finding confirmed and extended by experimental work. Work in Caenorhabditis elegans and Drosophila has highlighted the importance of phase 2 detoxification in extension of lifespan by reduced insulin/Igf-like signalling. Thorough characterization of systems for dietary restriction in C. elegans is starting to show differences in the mechanisms by which these interventions extend lifespan and has revealed a requirement for autophagy. The response to heat shock in C. elegans turns out to be systemic, and mediated by sensory neurons, with potentially interesting implications for the response of lifespan to temperature. Work in Escherichia coli and yeast has revealed a role for retention of aggregated proteins in the parent in the rejuvenation of offspring while, as in C. elegans, removal of the germ line in Drosophila turns out to extend lifespan. Aging research has suffered the loss of a great scientific leader, Seymour Benzer, and his trail-blazing work on aging and neurodegeneration is highlighted.     

A peroxiredoxin specifically expressed in two types of pharyngeal neurons is required for normal growth and egg production in Caenorhabditis elegans

A family of antioxidant proteins, the peroxiredoxins, serve two purposes, detoxification of reactive oxygen species and cellular signaling. Among the three peroxiredoxins of Caenorhabditis elegans (CePrx1-3), CePrx2 was found to have a very unusual expression pattern, restricted to only two types of pharyngeal neurons; namely, the single pharyngeal interneuron I4 and the sensory interneuron I2. CePrx1 and CePrx3-depleted worms showed no obvious phenotypic alterations, whereas worms devoid of CePrx2 were retarded developmentally and had a significantly reduced brood size. Other features, such as lifespan, pharyngeal activity or defecation rates were indistinguishable from those of wild-type worms. Recombinant CePrx2 revealed antioxidant activity, as it was able to detoxify hydrogen peroxide and butylhydroperoxide (t-BOOH), and to protect glutamine synthetase from inactivation by thiol-dependent metal-catalyzed oxidation. In addition, the molecule was able to act as a terminal peroxidase in the thioredoxin system. Expression of ceprx2 in C.elegans was induced after short-term exposure of worms to t-BOOH but survival of ceprx2 knockout mutants in the presence of reactive oxygen or nitrogen species was not impaired. Thus, CePrx2 may protect specifically the two types of neurons from oxidative damage or, more likely, plays a critical role in peroxide signaling in this nematode.     

The stn-1 syntrophin gene of C.elegans is functionally related to dystrophin and dystrobrevin

Syntrophins are a family of PDZ domain-containing adaptor proteins required for receptor localization. Syntrophins are also associated with the dystrophin complex in muscles. We report here the molecular and functional characterization of the Caenorhabditis elegans gene stn-1 (F30A10.8), which encodes a syntrophin with homology to vertebrate alpha and beta-syntrophins. stn-1 is expressed in neurons and in muscles of C.elegans. stn-1 mutants resemble dystrophin (dys-1) and dystrobrevin (dyb-1) mutants: they are hyperactive, bend their heads when they move forward, tend to hypercontract, and are hypersensitive to the acetylcholinesterase inhibitor aldicarb. These phenotypes are suppressed when stn-1 is expressed under the control of a muscular promoter, indicating that they are caused by the absence of stn-1 in muscles. These results suggest that the role of syntrophin is linked to dystrophin function in C.elegans.     

Glyoxalase-1 prevents mitochondrial protein modification and enhances lifespan in Caenorhabditis elegans

Studies of mutations affecting lifespan in Caenorhabditis elegans show that mitochondrial generation of reactive oxygen species (ROS) plays a major causative role in organismal aging. Here, we describe a novel mechanism for regulating mitochondrial ROS production and lifespan in C .  elegans: progressive mitochondrial protein modification by the glycolysis-derived dicarbonyl metabolite methylglyoxal (MG). We demonstrate that the activity of glyoxalase-1, an enzyme detoxifying MG, is markedly reduced with age despite unchanged levels of glyoxalase-1 mRNA. The decrease in enzymatic activity promotes accumulation of MG-derived adducts and oxidative stress markers, which cause further inhibition of glyoxalase-1 expression. Over-expression of the C .  elegans glyoxalase-1 orthologue CeGly decreases MG modifications of mitochondrial proteins and mitochondrial ROS production, and prolongs C .  elegans lifespan. In contrast, knock-down of CeGly increases MG modifications of mitochondrial proteins and mitochondrial ROS production, and decreases C .  elegans lifespan.     

Autophagy regulates ageing in C. elegans

The role of autophagy in ageing regulation has been suggested based on studies in C. elegans, in which knockdown of the expression of bec-1 (ortholog of the yeast and mammalian autophagy genes ATG6/VPS30 and beclin 1, respectively) shortens lifespan of the daf-2(e1370) mutant C. elegans. However, Beclin1/ATG6 is also known to be involved in other cellular functions in addition to autophagy. In the current study, we knocked down two other autophagy genes, atg-7 and atg-12, in C. elegans using RNAi. We showed that RNAi shortened the lifespan of both wild type and daf-2 mutant C. elegans, providing strong support for a role of autophagy in ageing regulation.     

A Na+/Cl- -coupled GABA transporter, GAT-1, from Caenorhabditis elegans: structural and functional features, specific expression in GABA-ergic neurons, and involvement in muscle function

GABA functions as an inhibitory neurotransmitter in body muscles and as an excitatory neurotransmitter in enteric muscles in Caenorhabditis elegans. Whereas many of the components of the GABA-ergic neurotransmission in this organism have been identified at the molecular and functional levels, no transporter specific for this neurotransmitter has been identified to date. Here we report on the cloning and functional characterization of a GABA transporter from C. elegans (ceGAT-1) and on the functional relevance of the transporter to the biology of body muscles and enteric muscles. ceGAT-1 is coded by snf-11 gene, a member of the sodium-dependent neurotransmitter symporter gene family in C. elegans. The cloned ceGAT-1 functions as a Na(+)/Cl(-)-coupled high-affinity transporter selective for GABA with a K(t) of approximately 15 microm. The Na(+):Cl(-):GABA stoichiometry for ceGAT-1-mediated transport process is 2:1:1. The transport process is electrogenic as evidenced from GABA-induced inward currents in Xenopus laevis oocytes that express ceGAT-1 heterologously. The transporter is expressed exclusively in GABA-ergic neurons and in two other additional neurons. We also investigated the functional relevance of ceGAT-1 to the biology of body muscles and enteric muscles by ceGAT-1-specific RNA interference (RNAi) in rrf-3 mutant, a strain of C. elegans in which neurons are not refractory to RNAi as in the wild type strain. The down-regulation of ceGAT-1 by RNAi leads to an interesting phenotype associated with altered function of body muscles (as evident from changes in thrashing frequency) and enteric muscles (as evident from the rates of defecation failure) and also with altered sensitivity to aldicarb-induced paralysis. These findings provide unequivocal evidence for a modulatory role of GABA and ceGAT-1 in the biology of cholinergic neurons and in the function of body muscles and enteric muscles in this organism.     

Otx/otd homeobox genes specify distinct sensory neuron identities in C. elegans

The mechanisms by which the diverse functional identities of neurons are generated are poorly understood. C. elegans responds to thermal and chemical stimuli using 12 types of sensory neurons. The Otx/otd homolog ttx-1 specifies the identities of the AFD thermosensory neurons. We show here that ceh-36 and ceh-37, the remaining two Otx-like genes in the C. elegans genome, specify the identities of AWC, ASE, and AWB chemosensory neurons, defining a role for this gene family in sensory neuron specification. All C. elegans Otx genes and rat Otx1 can substitute for ceh-37 and ceh-36, but only ceh-37 functionally substitutes for ttx-1. Functional substitution in the AWB neurons is mediated by activation of the same downstream target lim-4 by different Otx genes. Misexpression experiments indicate that although the specific identity adopted upon expression of an Otx gene may be constrained by the cellular context, individual Otx genes preferentially promote distinct neuronal identities.     

A new putative cyclic nucleotide-gated channel gene, cng-3, is critical for thermotolerance in Caenorhabditis elegans

Cyclic nucleotide-gated (CNG) channels encoded by tax-4 and tax-2 genes are required for chemo- and thermo-sensation in Caenorhabditis elegans. Here we report the identification and the characterization of cng-3, a new CNG channel gene, found in C. elegans. CNG-3 contains six putative transmembrane regions and a cyclic nucleotide-binding domain that show high homology with CNG channels of higher animals as well as TAX-4. The expression of cng-3 is detected from early stages in worm development and restricted in five sensory neurons of amphid including AFD neuron. While a cng-3 null mutant displays normal chemotaxis to volatile odorants, the mutant worms exhibit impaired thermal tolerance. These results indicate that CNG-3, a new member of CNG channel subunits, may play a critical role in sensation or response of thermal stress in C. elegans.     

SIRT1与基因转录

 SIRT1（silent mating type information regulation 2 homolog 1）是一种具有NAD-依赖的蛋白去乙酰化酶活性的多功能转录调节因子.在体内通过对几种控制代谢及内分泌信号的转录因子去乙酰化作用来调节其活性.从而广泛参与调控哺乳动物细胞寿命的不同信号通路及糖代谢,胰岛素分泌等多条代谢通路,预示着SIRT1在医学临床应用和研究中可能极具应用价值.     

Caenorhabditis elegans as a screening tool for the endothelial cell-derived putative aging-related proteins detected by proteomic analysis

Endothelial cells go through progressive pathophysiologic modification as cellular senescence progresses. In vitro, endothelial cell senescence is accompanied by failure of proliferation and by perturbations in gene and protein expressions. Moreover, this cellular senescence in culture has been proposed to reflect processes that occur in the organism in vivo and free radical theory is accepted to be the most plausible explanation for this process. We have screened proteins involved in both cellular senescence and reactive oxygen species induced condition using 2-D gel analysis and found that ubiquitin carboxyl terminal hydrolase L1, peroxyredoxin 2, peroxyredoxin 4, fatty acid binding proteins (FABPs), and 5'-AMP-activated protein kinase beta-1 subunit were candidate aging-related proteins. To evaluate in vivo function of these proteins, Caenorhabditis elegans (C. elegans) knock-down system using RNA interference was applied. Aging-specific expression of lipofucsin and the lifespan of knocked-down C. elegans were observed to assess the outcome. Interestingly, the inhibition of the genes led to short lifespan and earlier accumulation of lipofucsin with increasing age when compared with the wild type. These results suggest that the above genes may be related to cellular senescence process in determining the longevity in C. elegans and that gene inactivation renders animals susceptible to oxidative stress.